The determination of the presence or amount of substances is commonly performed by immunoassay. Immunoassay techniques are based on the binding of the antigenic substance being assayed (the "target analyte") to a receptor for the target analyte. The term "analyte" or "target analyte" refers to the compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor. Immunoassay methods can be applied to the assay of many different biologically active substances. Among such substances are haptens, hormones, gamma globulin, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and animal venom, and many drugs. Similar techniques can be used in non-immunological systems with, for example, specific binding proteins.
Many different types of immunoassays are known in the art, including competitive inhibition assays, sequential addition assays, direct "sandwich" assays, radioallergosorbent assays, radioimmunosorbent assays and enzyme-linked immunosorbent assays. The basic reaction underlying most immunoassays is the binding of certain substance, termed the "ligand" or "analyte", by a characteristic protein (receptor) to form a macromolecular complex. These binding processes are reversible reactions, and the extent of complex formation for particular analyte and receptor concentrations is regulated by an equilibrium constant according to the law of mass action. Thus, at equilibrium, some of the analyte always exists unbound (free).
The measurement of target analytes in biological fluids, such as serum, plasma and whole blood, requires immunoassay methods which are both specific and sensitive. Both the specificity and sensitivity of an immunoassay depend on the characteristics of the binding interaction between the target analyte and the receptor involved. For example, the reaction must be specific for the analyte to be measured and the receptor used should not bind to any other structurally related compounds. In addition, by choosing a receptor with a high affinity for the target analyte, the sensitivity can be increased. The label used to monitor the assay also affects the sensitivity of an immunoassay. Either the target analyte or the receptor may be labeled to permit detection. Labels currently used for immunoassay of target analytes in biological fluids include radioisotopes (radioimmunoassay, RIA), enzymes (enzyme immunoassay, EIA), fluorescent labels (fluorescence immunoassay, FIA), and chemiluminescent labels (chemiluminescent immunoassay, CIA). Theoretically, fluorometry is capable of being one of the most sensitive of all analytic tools as it is possible to detect single photon events.
FIAs use fluorescent molecules as labels and may be either heterogeneous or homogeneous. Fluorescent molecules (fluorophores) are molecules which absorb light at one wavelength and emit light at another wavelength. See Burd, J. F., "Fluoroimmunoassay--Application to Therapeutic Drug Measurement," in P. Moyer et al., Applied Therapeutic Drug Monitoring, American Association of Clinical Chemistry (1984). Typically, an excitation pulse of radiation is directed onto or into a sample, followed by fluorescence of the sample, and the detection of the fluorescence radiation. Fluorescence is a phenomenon exhibited by certain substances, which causes them to emit light, usually in the visible range, when radiated by another light source. This is not reflection, but creation of new light. Current commercially available assay methods use fluorescein, which emits green light when radiated by a light source containing blue light. In addition to fluorescing, fluorescein (and other fluorophores) emit polarized light. That is, the light emitted has the same direction of polarization as the incident polarized light, if the fluorescein molecule is held fixed with its transition moment parallel to the electric field of the excitation.
Using a fluorescent polarizing probe in a competitive binding immunoassay provides a type of FIA called a fluorescence polarization immunoassay (FPIA). In this type of assay, the smaller the molecule is, the smaller its rotational relaxation time and the faster it rotates. Typically, antibody molecules are much larger than drug or drug-probe molecules. One advantage of the polarization technique is the elimination of a step to separate unbound probe. Although the unbound tracer is not physically eliminated from the samples in FPIA, its contribution is readily assessed by the polarization. Another advantage in the FPIA technique is lack of dependence on intensity. Unlike most assays using a light measurement, in which it is the intensity of the light that is correlated to drug concentration (so any variations in source light intensity will directly affect the sensitivity of the assay), the sensitivity of FPIAs is independent of intensity variations. Conventional FPIAs require separate measurements of both blank and sample.
One problem which has plagued fluorescence immunoassays has been discriminating the fluorescent signal of interest from background radiation. The intensity of signal from background radiation may be up to 10,000 times larger than the intensity of the fluorescent signal of interest. The problem of background detection is particularly pronounced in assay of biological samples. Many of the current fluorescence assays use the fluorescent molecule, fluorescein. Fluorescein has an excitation maximum of 493 nm, and there are numerous substances in biological fluids with overlapping excitation and emission similar to fluorescein. For example, in the analysis of blood plasma, the presence of a naturally occurring fluorescable material, biliverdin, causes substantial background radiation. Such compounds are highly fluorescent and contribute significant background signals which interfere with the label's signal, thus limiting the sensitivity of assays using fluorescein labels.
Two factors commonly viewed as going hand in hand and contributing to the problem background radiation are: (1) solvent sensitivity and (2) non-specific binding. Solvent sensitivity refers to tendency of a solvent to affect the fluorescent signal of the dye. For example, several dyes that are fluorescent in organic solvents tends to aggregate in agneous solutions and therefore exhibit quenched fluorescence. Non-specific binding refers to the tendency of sample components to interfere with the fluorescent signal of the dye. For example, serum components such as HSA often bind to conventional fluorescent dyes and quench or enhance interfere with the fluorescent signal.
It has been recognized that for analysis of biological fluids, it would be desirable to use a dye or label which is excitable at radiations of wavelengths of greater than background radiation. However, even though the background fluorescence of serum falls off at wavelengths approaching 600 nm, significant interference persists until 650 nm or greater. Previous attempts to create dyes of such wavelengths have been unsuccessful. See, e.g., Rotenberg, H. and Margarfit, R., Biochem. Journal 229:197 (1985); and D. J. R. Laurence, Biochem. Journal 51:168 (1952). Fluorescent dyes having emission wavelengths which reduce interference from background fluorescence include cyanines, porphyrins and azaporphyrins. However, it has been found that the use of such labels in fluorescence assays is limited by the problems of solvent sensitivity (significantly decreased fluorescence intensity in the aqueous assay solution in comparison to dimethylformamide) and non-specific binding to biological materials (significantly decreased fluorescence intensity in purified or isolated sample comparison to a sample containing serum components such as HSA).
Use of fluorophores having long decay times is especially important in techniques such as transient state assays where there is a need for fluorophores whose emissions may be measured over a time period of up to about 20 nanoseconds. It has been found that for fluorophores natural lifetime and extinction coefficient vary antibatically (i.e., when one increases, the other decreases; although they need not change at rates inversely to each other). Also, fluorophores having longer fluorescent lifetimes are more apt to be deactivated. Accordingly, fluorophores having enhanced decay times, i.e. having decay times which approach their natural lifetime, offer greater quantum yields and, thus, greater sensitivity.
Earlier attempts to overcome the problem of background radiation have involved: (1) the use of filters to reject detected radiation at all but a narrowly defined wavelength band; (2) binding fluorescent probes to a macro-molecular support thereby minimizing the effect of serum proteins while allowing detection of the binding to a target analyte, (see U.S. Pat. No. 4,615,986, issued Oct. 7, 1986); and (3) the use of chemical groups designed to improve the functional properties of the dyes (see Mujumdar, et al. Bioconjugate Chemistry 4: 105-110, 1993).
Arnost et al., U.S. Pat. No. 4,886,744, issued Dec. 12, 1989 ("Arnost") describes a fluorescent label with an unsymmetrical cyanine dye of structure: ##STR1##
Certain of the R groups can be "a hydrophilic group". Hydrophilllic groups are defined as including a variety of possibilities, including a polyether, in order to decrease aggregation and non-specific binding. The data set forth by Arnost are said to show that some of the "hydrophilic groups" decrease the nonspecific binding somewhat and lowered the binding constants from 7E5 down to 1.5E4, a factor of less than 50. The components of Arnost, et al. typically have an excitation maximum of less than 500 nm.